Program Director/Principal Investigator (Last, First, Middle): Engel, Joanne 2R01 AI042806-11A1 ABSTRACT Pseudomonas aeruginosa (PA) is one of the most virulent opportunistic pathogens of man. The morbidity of PA infections results from the ability of the bacterium to colonize previously injured epithelium and prevent its repair, leading to further tissue damage and dissemination. Our initial work was based on a novel genetic screen in mammalian epithelial cells that identified new virulence factors of PA required for epithelial cell injury, including the type III secreted toxin ExoU. We demonstrated that the type III secretion system (T3SS) contributed to virulence of acute PA infections, both in tissue culture, animal models, and in retrospective human studies. Our screen also identified new regulators of type IV pili (TFP), including the Chp gene cluster and FimL. During the most recent funding period (2003-current), we have shown that both domains of ExoT, a T3SS effector encoded by almost all strains of PA, contributes to the biological activities of ExoT in tissue culture models of wound healing and in mouse models of acute infection. In addition to its inhibitory effects on bacterial internalization, cell migration, and host cell cytoskeleton, we have discovered that ExoT inhibits cytokinesis and induces apoptosis, in part through interfering with Crk function ). These studies have led to a previously unappreciated role of Crk in cytokinesis and will open up new areas of research in the field of cell division. We have discovered that Cbl-b, an adaptor molecule with E3 ubiquitin ligase activity modulates the stability of ExoT by targeted proteasomal degradation and limits the dissemination of P. aeruginosa in murine models of infection. These results demonstrate a previously unappreciated role for Cbl-b in early host defense response and provide the first example of an animal host gene that is required for the in vivo resistance to disease mediated by a T3SS effector. We have investigated the role of the Chp operon and FimL in regulating twitching motility and T3SS. Finally, we have used forward genetics to carry out a genome-wide screen using RNAi-mediated gene inactivation in Drosophila S2 cells to identify host factors required for T3SS-dependent intoxication by ExoU. In the next granting period, we will our focus on understanding (i) the mechanism by which FimL regulate diverse virulence circuits including TM and T3SS (ii) the role of Cbl-b in limiting ExoT-mediated damage in in vivo models of infection and (iii) the host cell factors required for ExoU- and T3SS-mediated damage. This analysis has the potential to yield new insights into the pathogenesis of acute PA infections, enhance our understanding of how the well conserved and critically important T3SS of gram negative pathogens subverts key host cell processes, and identify novel targets for therapeutic, preventative, or diagnostic strategies. It opens up the possibility of targeting host cell processes to treat infectious diseases.